Hepatitis C virus (HCV) is an example of a virus that uses an internal ribosome entry site (IRES) RNA to drive production of all the viral proteins. The HCV IRES RNA forms a specific structure that binds directly to the 40S ribosomal subunit and in so doing changes the conformation of the subunit. This physical manipulation of the ribosome appears to be a critical component of the mechanism by which the HCV IRES hijacks the translation machinery. However, how the IRES accomplishes these structural changes, how they propagate through the ribosome, and how the induced structures compare to canonical ribosome conformations remains unknown. In addition, there is growing evidence that the HCV IRES is one component of the virus' strategy to evade the host cell's innate immune response, in part by driving translation initiation by alternate, eukaryotic initiation factor (eIF) 2-independent pathways operating during cellular stress. While the mechanism by which the HCV IRES operates in unstressed cells is well-studied, the pathway used in stressed cells is very poorly understood. We seek to explore the ability of the IRES to manipulate the host cell's machinery by accessing alternate translation initiation pathways and also through physical manipulation of the ribosome. We propose two aims: 1) Define the IRES domains, factors, and mechanisms that enable the HCV IRES to operate under conditions of cellular stress, and 2) Determine the high-resolution structure of an HCV IRES RNA bound to a ribosome or ribosomal subunit. To accomplish these aims, we will use a combination of biochemistry, cell culture-based assays, and structural biology in an integrated approach focused on developing new models for HCV IRES function at an unprecedented level of detail. Several of the methods and tools we will use have been developed in our lab and thus are unique to our lab. Our studies promise to contribute new discoveries to how IRESs function, how mammalian ribosomes operate, how ribosomes can be manipulated, and how translation occurs during periods of cell stress.